Recombinant protein comprising starch binding domain and use thereof

ABSTRACT

A recombinant protein is prepared comprising a polypeptide of interest and a starch binding domain (SBD). The said SBD is obtainable from glucoamylase of fungi genus  Rhizopus . The said recombinant protein comprising the said SBD can be purified by contacting with an affinity matrix such as starch, the SBD binds to the affinity matrix to isolate the recombinant protein. The recombinant protein can be purified by separating the association between the SBD and the affinity matrix by acid, alkaline, salt, or sugar. The polypeptide of interest may be an antibody, an antigen, a therapeutic compound, an enzyme, or a protein and may apply in pathogen destruction, vaccine producing, and oral care product manufacturing. The SBD further provides as a tool to screen or identify polysacchrides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of the pending U.S. patent applicationSer. No. 11/070,271 filed on Mar. 3, 2005, all of which is herebyincorporated by reference in its entirety.

Although incorporated by reference in its entirety, no arguments ordisclaimers made in the parent application apply to this divisionalapplication. Any disclaimer that may have occurred during theprosecution of the above-referenced application(s) is hereby expresslyrescinded. Consequently, the Patent Office is asked to review the newset of claims in view of the entire prior art of record and any searchthat the Office deems appropriate.

FIELD OF THE INVENTION

This invention relates to functions of starch binding domain (SBD) ofglucoamylase in fungi Rhizopus spp. A recombinant protein comprising theSBD can be produced and purified by using the SBD as a tag. Thisinvention further relates to a novel method and a kit to purify therecombinant protein and novel applications of the SBD and therecombinant protein.

BACKGROUND OF THE INVENTION

Production of proteins by expression in microbial systems has become asignificant source of high value, medically important proteins.Purification and recovery of recombinant proteins are majorconsiderations in the design of a fermentation process. Whiletraditional methods of protein purification can be used to isolate aproduct, improved methods include the use of recombinant proteins.Recombinant proteins can be purified by affinity chromatography, thedesired component of the recombinant protein being purified by virtue ofits covalent attachment to a polypeptide, which binds to an affinitymatrix.

Certain systems exist for isolating proteins by the principle ofaffinity chromatography.

U.S. Pat. No. 5,643,758 describes a system comprising maltose-bindingprotein (MBP). A cloned gene is inserted into a pMAL vector down-streamfrom the malE gene, which encodes MBP. The vector is transformed to ahost cell, then the recombinant protein can express in the host cell.The cell lysate or media fraction is loaded to a column containingaffinity matrix amylose and washed several times, then using a largeamount of maltose to elute the recombinant protein.

U.S. Pat. No. 5,202,247 describes a system comprising cellulose-bindingdomain. A cellulose column can be used to purify the recombinant proteinwhich contains cellulose-binding domain. The cell lysate or mediafraction is loaded to the column and washed. The interaction betweencellulose-binding domain and cellulose appears to be driven byhydrophobic interaction at neutral pH. The general method for elutionused low polarity solvents such as ethlylene glycol, prior to removal ofthe low polarity solvents by dialysis and filtration.

A chitin-binding domain and an inducible-splicing linker region can befused in the C-terminus or N-terminus of a target protein. The celllysate or media fraction is loaded to the column and washed. Thechitin-binding domain binds to the chitin column to immobilize therecombinant protein. In the presence of thiols such as DTT or cysteine,the linker region undergoes specific self-cleavage which releases thetarget protein from the chitin-bound chitin-binding domain.

These current protein purification systems have some disadvantages. Thepurification processes are inconvenient and laborious. The columns usedin purification are expensive. Limitations for protein purification ofthese systems include unable to isolate the recombinant protein incertain conditions such as EDTA-containing samples as well as thecurrent protein tags being used are relatively large as compared to thetarget protein bigger than that of this invention.

SUMMARY OF THE INVENTION

This invention provides a starch binding domain comprising acharacteristic dissociation constant (K_(d)) of 0.5˜2.29 μM. Thisinvention also provides a recombinant protein comprising a polypeptideand a starch-binding domain of the invention. This invention alsoprovides an expression vector comprises a gene encoding starch bindingdomain of the invention. This invention also provides a host celltransformed or transfected with a vector for the replication andexpression of DNA encoding the recombinant protein comprising apolypeptide of interest and a starch-binding domain of the invention.This invention further provides a method for purifying a recombinantprotein comprising a polypeptide and a starch binding domain of theinvention from a biological liquid, which method comprises: (a) applyingthe biological liquid containing the recombinant protein directly to anaffinity matrix; (b) eluting the affinity matrix by eluent; and (c)dialyzing the eluent. This invention further provides a kit forpurifying a recombinant protein comprises an expression vector used toexpress the recombinant protein. This invention further provides amethod of sorting carbohydrate-containing molecule in a samplecontaining various carbohydrate-containing molecules comprises: (a)preparing a set of separator with different K_(d) values of carbohydratebinding domain; (b) putting the sample into the set of separator; and(c) sorting the molecule into bound and unbound groups in accordancewith different K_(d) values.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates purification of SBD-6H using starch. The purifiedSBD-6H is subjected to SDS/PAGE (15% gel) and the gel is stained withCoomassie Brilliant Blue R-250 for protein detection. Lane 1, molecularmass standards (molecular masses indicated at the left); lane 2, Twomicrogram of SBD-6H that is purified on corn starch.

FIG. 2 illustrates effects of pH on binding ability and stability. Theeffects on binding ability () and stability (◯) of the SBD-6H atvarying pH are measured as described in the Experimental section. Therelative binding ability of SBD-6H assayed in pH 5 is normalized as100%.

FIG. 3 illustrates adsorption rate of SBD-6H with insoluble starch. Theassociation rate of SBD is analyzed by applying SBD-6H at differentconcentrations (▪, 15.6 μM; ▴, 22.5 μM; , 27.2 μM) to corn starch for 5h. Bound protein is calculated from the difference between the initialand unbound protein concentrations at different time points to determinethe association rate.

FIG. 4 illustrates K_(d) and B_(max) determination of the SBD-starchinteraction. Starch binding assays are performed with granule cornstarch at different protein concentrations. The K_(d) and B_(max) aredetermined by fitting to the non-linear regression of the bindingisotherms for one binding site saturation binding.

FIG. 5 shows construction of SBD-tagged fusion proteins. eGFP: geneencoding green fluorescent protein; SBD: starch binding domain; Km^(r):kanamycin resistance gene.

FIG. 6 illustrates purification of SBD-tagged fusion proteins usingstarch. The purified fusion protein is subjected to SDS/PAGE (15% gel)and the gel is stained with Coomassie Brilliant Blue R-250 for proteindetection. Lane 1, molecular mass standards (molecular masses indicatedat the left), lane 2, soluble fraction, and lane 3, elution. ArrowheadI: SBD-eGFP, arrowhead II: eGFP, and arrowhead III: SBD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is provided with a specially high affinity andstrong binding ability of a starch binding domain (SBD) of an enzymeglucoamylase from fungal genus Rhizopus, especially in Rhizopus oryzae.The glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) is amultidomain exo-acting glycoside hydrolase that catalyzes the release ofβ-D-glucose from the non-reducing ends of starch and related substrates.Glucoamylase is a modular protein comprising a catalytic domain (CD),and a starch binding domain (SBD), categorized to carbohydrate-bindingmodules (CBMs). The two independent domains are connected with anO-glycosylated linker (for detailed information, see URLhttp://afmb.cnrs-mrs.fr/CAZY/index.html; Boraston et al. Biochem J(2004) 382: 769-781; Fang et al. Protein Engineering (1998) 11:119-126;Sauer et al. Biochemistry (2001) 40: 9336-9346).

The CBMs mediate the interaction of glycoside hydrolases with substratesby concentrating the catalytic domains on the surface of insolublepolysaccharides (Bolam et al. Biochem J (1998) 331:775-781). Thesignificant decreases in the activity of enzymes on insoluble but notsoluble polysaccharides are observed by both proteolytic excision andtruncation of CBMs from glycoside hydrolases (Charonck et al.Biochemistry (2000) 39:5013-5021; Ali et al. Biosci Biotechnol Biochem(2001) 65:41-47). Different from all C-terminal SBDs belonging to theCBMs family 20, the N-terminal SBDs of glucoamylases are classified asmembers of CBM family 21. At present, glucoamylases from Rhizopusoryzae, Arxula adeninivorans and Mucor circinelloides have the CBMfamily 21 starch binding domain located at the N-termini(Houghton-Larsen et al. Appl Microbiol Biotechnol (2003) 62: 210-217).

Comparison of the amino acid sequences of SBDs from these species and A.niger SBD shows that the homology between R. oryzae SBD and that of A.adeninivorans, M. circinelloides, and Aspergillus. niger is 35.4%, 67.3%and 26.1%, respectively. The difference of the protein sequences betweenCBM 20 and CBM 21 families implies that these SBDs are distinct inbiological and biochemical functions.

The polypeptide of the SBD according to the invention can be obtainablefrom Rhizopus spp. The embodiment in this invention is Rhizopus oryzae.The amino acid sequences of SBDs obtainable from Simpson strain, wildtype strain and a strain in U.S. Pat. No. 4,863,864 as shown in SEQ IDNO. 1, 2, and 3 are different from each other. The SBDs of Rhizopusoryzae including allelic variation and derivatives have high affinitywith starch and express strong starch-binding ability as compared withknown SBDs.

In this invention, the DNA molecule encoding SBD is amplified by PCR.The PCR product is cloned into an expression vector and transformed intoa host cell. Transformed host cells are induced to express SBD. Celllysate is harvested and the supernatant is applied directly to theaffinity matrix, then washed and eluted. The elution buffer can be salt,sugar, and/or acidic or alkaline. The SBD of Rhizopus spp. is firstcharacterized in this invention to be stable in a wide range of pH anddissociate with starch in acidic or alkaline environments. In thisinvention, it is first identified that the dissociation constant (K_(d))value of SBD in Rhizopus spp is lower than that of known species such asAspergillus niger, and the starch binding ability of SBD in Rhizopus sppis much higher than that of known species such as Aspergillus niger. Thepreferred embodiment of the dissociation constant (K_(d)) value of SBDsin Rhizopus to granular corn starch is 0.5˜2.29. The more preferredembodiment of the dissociation constant (K_(d)) value is 1.0˜2.0. Thefurther preferred embodiment of the dissociation constant (K_(d)) valueis 1.3˜1.6. The most preferred embodiment of the dissociation constant(K_(d)) value is 1.43.

This invention also provides a recombinant protein comprising a SBD ofthis invention and a polypeptide of interest. A gene encoding apolypeptide of interest is cloned into the SBD-expression vector aspreviously described. The cloning site is neighboring the sbd gene,either upstream or downstream of sbd gene. The SBD can be linked withthe polypeptide in N-terminus or in C-terminus. This fusion geneexpression vector can be transformed into a host cell, includingbacteria, yeast, fungi, mammalian cell, or insect cell. The recombinantprotein can be expressed in the transformed cell. Thus, this recombinantprotein according to this invention can be purified by using starch asaffinity matrix, via the association between SBD and starch.

The affinity matrix used in recombinant protein purification can be acomponent recognized by SBD. The SBD according to this invention canbind the glucose-glucose linkage structure, both α-1,4- andα-1,6-linkage. By this characteristic, the affinity matrix can be acomponent containing the formula:

(X-X)_(n)

X means glucose molecule, the linkage between glucose and glucose isα-1,4-linkage or α-1,6-linkage and n is 1 or more than 1. One componentcontains the former structure in any part structure such as main chain,side chain, or modified residue can be selected to be affinity matrix.For example, starch, mannose, dextran, or glycogen can be affinitymatrix.

This invention provides a recombinant protein purification method byusing a starch binding domain of the invention and an affinity matrix.The method comprises (a) applying the biological liquid containing therecombinant protein directly to an affinity matrix; (b) eluting theaffinity matrix by elution buffer; and (c) dialyzing the eluent. Thisinvention can be used to increase the production, activity, stability orsolubility of recombinant proteins and polypeptides. This invention issuitable for large-scale protein purification with wide range of optimalpH. The yield of the purified recombinant protein is high withsufficient purity (greater than 95%). The advantages of this inventionalso includes that various elution buffers and affinity matrices can bechosen and be available commercially, the former includes sugar, salt,and/or pH and the latter includes starch, mannose, dextran, or glycogen.Further, SBD is a smaller tag than commonly used fusion protein tagsincluding glutathione S-tansferase (GST), MBP, thioredoxin (Trx), orNus, and this system is appropriated to purify particular samplescomprising EDTA-, EGTA-, or DTT-containing sample.

This invention provides a kit to purify a recombinant protein andapplications thereof. The kit comprises an expressing vector to expressthe recombinant protein according this invention. The kit furthercomprises an affinity matrix and an elution buffer. The affinity matrixcan bind SBD to isolate the recombinant protein. The elution buffer isused to separate the association between SBD and affinity matrix.

The polypeptide of interest as previously described may be an antibody,an antigen, a therapeutic compound, an enzyme, or a protein. Via the kitprovided by this invention, these products can be over-produced andpurified rapidly. The applications of these products are described asfollows.

The polypeptide of interest may be an antibody or a therapeuticcompound, which targets and allows damage/destruction or detection ofthe pathogen. This invention allows the antibody or the therapeuticcompound easily purified by using starch and can be further isolated bycleaving the SBD region. The purified product can be used for therapy ordetection of pathogens.

The polypeptide of interest may be an antigen or an antigenic compound,which can induce immune responses. This fusion protein can bind to anaffinity matrix such as starch. The fusion protein-bound starch can besupplied as source of foods. When eating the fusion protein-boundstarch, users will gain vaccination effect. The fusion protein-boundedstarch can be an oral vaccine.

This invention further provides an application relates an oral carecomposition. The oral care composition is comprising the SBD of theinvention, wherein said composition is selected from the groupconsisting of a toothpaste, dental cream, gel or tooth powder, odontic,mouthish, pre- or post brushing rinse formulation, chewing gum, lozenge,and candy. The oral care composition further comprises a fusion productbetween one or more SBDs and an enzyme selected from the groupconsisting of oxidases, peroxidases, proteases, lipases, glycosidases,lipases, esterases, deaminases, ureases and polysaccharide hydrolases.This oral care product can digest oral polysaccharide and preventformation of dental caries.

This invention further provides a method for sortingcarbohydrate-containing molecules by using different K_(d) values ofcarbohydrate binding domains. The carbohydrate-containing molecules maybe a mixture of various glycoproteins, and the carbohydrates may bemonosaccharide, disaccharide, or polysaccharide. For example, the userscan sort glycoproteins by using starch-binding domains (SBDs) as a setof separator; maybe a column; with different K_(d) values to generate aseries of matrices with gradient ligand binding abilities. The SBDs maybe obtainable from Rhizopus spp according to this invention, includingwild type and mutants. The SBDs may be obtainable from the species whichis known to have SBDs such as CBM family 21 and CBM family 20. Theseknown species include Arxula, Lipomyces, Aspergillus, Bacillus,Clostridium, Cryptococcus, Fusarium, Geobacillus, Neurospora,Pseudomonas, Streptomyces, Thielavia, and Thermoanaerobacter.

Loading the sample into the set of separator, the SBD-bound moleculesare kept in the column and the unbound molecules are excluded. Thus, thesample can be sorted into SBD-bound and unbound groups. Collectingunbound molecules in one container, and eluting SBD-bound molecules inanother container, both can be further sorted. The further sortingmethods may be based on chemical, physical or biological propertiesincluding liquid chromatography analysis, mass spectrometric analysis,lectin immunosensor techniques, two-dimensional gel electrophoresis,enzymatical techniques, and chemical derivatizations.

EXAMPLE

The following examples are offered by way of illustration and not by wayof limitation.

Example 1 (A) Construction of SBD-6H

The gene encoding glucoamylase SBD from residues 26-131 was amplified bypolymerase chain reaction (PCR) using the forward primer5′-CATATGGCAAGTATTCCTAGCAGT-3′ and the reverse primer5′-CTCGAGTGTAGATACTTGGT-3′ (restriction sites, shown in bold, wereincorporated into the two primers). The PCR product was cloned into thepGEM-T Easy cloning vector (Promaga) and verified by DNA sequencing. Thesbd gene fragment was subsequently ligated into pET23a(+) expressionvector (Novagen) at NdeI and XhoI sites to generate pET-SBD. SBD-6Hencoded by pET-SBD contains a C-terminal His₆ tag. The construct wastransformed into competent Escherichia coli BL21-Gold (DE3) (Novagen)for protein expression.

(B) Expression and Purification of SBD-6H

BL21-Gold (DE3) cells transformed with pET-SBD were grown in LB mediumcontaining 100 μg/ml ampicillin at 37° C. until the OD₆₀₀ reached 0.6.The temperature was then reduced to 20° C., and isopropylβ-D-thiogalactoside (IPTG) was added to a final concentration of 400 μMto induce recombinant protein expression. After further incubation for16 h, cells were harvested by centrifugation at 3,700 g for 15 min at 4°C., the resultant pellet was resuspended in 20 ml of binding buffer (50mM sodium acetate, pH 5.5) and then homogenized (EmulsiFlex-05homogenizer). The cell debris was removed by centrifugation at 16,000 gfor 15 min at 4° C. The supernatant was applied directly to the granularcorn starch (pre-washed with binding buffer) and then incubated at 25°C. for 30 min with gentle shaking. The starch was washed with 10 columnvolumes of binding buffer twice and then eluted with 2 column volumes ofelution buffer (50 mM glycine/NaOH, pH 10). The solution was dialyzedagainst binding buffer employing an Amicon stirred-cell concentrator(Millipore) equipped with a PM-10 membrane (10 kDa cut-off). Eachfraction was analyzed by 15% SDS-PAGE gel and then stained withCoomassie brilliant blue. The protein concentrations were assayed bybicinchoninic acid (BCA) reagent kit (Pierce).

SBD-6H fused was expressed in E. coli BL21-Gold (DE3) at an optimizedcondition. After induction period, the soluble fraction was one-steppurified by granular corn starch. Since low elution efficiency of SBDwith maltose as elution agent employing previously reported methods wasobserved (Paldi et al. Biochem J (2003) 372: 905-910), here a substituteprocedure for the elution of SBD with glycine/NaOH (pH 10) was adopted.Purified SBD-6H was analyzed by 15% SDS-PAGE and stained with Coomassiebrilliant blue as shown in FIG. 1. SBD-6H derived from the signal steppurification was homogeneous. This modified method enables fast andefficient purification of SBD from bacterial lysate with high purity(>98%) and yield (>90%).

Example 2 Effect of pH on Binding Ability and Stability

For the measurement of binding ability, SBD-6H and granular corn starchwere incubated in buffers with varied pH values for 1 h as described inthe starch binding assay. The stability was measured by keeping SBD-6Hat 25° C. for 30 min in different buffers including glycine/HCl (pH 3),sodium acetate/acetic acid (pH4-5), Na₂HPO₄/NaH₂PO₄ (pH 6-7), Tris/HCl(pH 8) and glycine/NaOH (pH 9-10). The remaining binding ability wasmeasured at 25° C. for 1 h in sodium acetate buffer (50 mM, pH 5.5). Therelative binding ability of SBD-6H assayed at pH 5 was normalized as100%.

The stability of SBD-6H was analyzed in the pH range from 3 to 10 and itindicated that the SBD-6H was stable in a wide pH range, even at theextreme acidic and alkaline conditions (FIG. 2). The binding assays wereused to identify the optimal pH range of SBD-6H on adsorption ofinsoluble starch. As shown in FIG. 2, the pH range for maximal SBD-6Hbinding was 5 to 6, whereas relative binding ability at pH 4 and pH 8was only 60.6% and 55.1%, respectively.

As shown in FIG. 2, SBD-6H was stable in a broad pH range, even at theextreme of the acidic and alkaline pH. It also indicated that thebinding ability of SBD to starch was pH-dependence. The optimal pH forbinding was around 5 to 6 similar to previous studies used maltose torelease the SBD from starch even though maltose was not the optimalelution agent. Here, we had overcome this problem based on theproperties of the pH-dependent binding ability. The elevated elutionefficiency (>90%) was performed using an alkaline buffer as an elutionagent.

Example 3 Association Rate of SBD-6H to Insoluble Starch

The purified SBD-6H in the concentration range from 15.6 to 27.2 μM wasadded to 1 mg/ml of pre-washed granular corn starch and incubated at 25°C. with gentle stir for 5 h. The binding was terminated at differenttime intervals by sedimentation of the starch. After centrifugation at16,000 g for 10 min at 4° C., the protein concentration of thesupernatant (unbound protein) was determined by the BCA assay, and theamount of bound protein was calculated from the difference between theinitial and unbound protein concentrations. The bound protein atequilibrium expressed as micromole of protein per gram of starch was alinear function of the free (unbound) protein in the range of proteinconcentrations assayed.

The adsorption kinetics of SBD-6H to granular corn starch was performedat different protein concentrations as shown in FIG. 3. About 50% of theSBD-6H was bound to the insoluble starch during the first 10 min(relative to the amount of bound protein at equilibrium state). In theinitial period (up to 40 min), the adsorption exhibited a linear phase.The amount of bound proteins was in proportion to the incubation time.Similar association rates (0.18±0.1 μmol/min·g), indicated by theinitial slope of the binding curves at different SBD-6H concentrationswere observed. After the linear phase (after 40 min), the bound proteinincreased slowly within prolonged incubation time. At the time of 120min, more than 95% relative binding was reached. The time required forreaching the equilibrium was similar to Aspergillus SBDs (Paldi et al.Biochem J (2003) 372: 905-910).

Example 4 Starch Binding Assay

The starch-binding isotherm was analyzed as saturation binding assay:the SBD-6H was mixed with 1 mg/ml of pre-washed granular corn starch andincubated at 25° C. with gentle stir for 16 h. After centrifugation at16,000 g for 10 min at 4° C., the amount of bound protein wascalculated. Controls with protein but no starch were included to ensurethat no precipitation occurred during the period of assay. Thedissociation constant (K_(d)) and the maximal amount of bound protein(B_(max)) were determined by fitting to the non-linear regression of thebinding isotherms, and equation (1) was used for one binding sitesaturation binding.

B=B _(max) F/(K _(d) +F)  (1)

where B (μmol) represents the bound protein; B_(max) (μmol) was themaximal amount of bound protein; F (μmol) was the free protein in thesystem and K_(d) (μmol) was the equilibrium dissociation constant. Theunits of the B_(max) and K_(d) values calculated were converted intoμmol/g and μM, respectively.

SBD-Binding Affinity and Capacity

Starch-binding isotherm as shown in FIG. 4 was used to study theaffinity and capacity of purified SBD-6H. Equation (1) was used tocalculate the non-linear regression curves of the equilibrium isothermand determine the binding parameters (B_(max) and K_(d)). The bindingparameters, K_(d) and B_(max), determined by a two-parameter model forone binding site saturation binding, were 1.43±0.14 μM and 41.14±1.05μmol/g, respectively. In addition, the dissociation constants from theengineered and proteolytically produced Aspergillus SBDs (3.2±0.9 μM and12.7±0.5 μM respectively) were approximately 2-10 times higher than thatof SBD-6H (Paldi et al. Biochem J (2003) 372: 905-910; Belshaw et al.FEBS Lett (1990) 269: 350-353). These observations indicate that SBD-6Hhas better binding affinity to insoluble starch than Aspergillus SBD. Onthe other hand, the B_(max) values of the engineered and proteolyticallyproduced Aspergillus SBDs (0.56 μmol/g and 1.08±0.02 μmol/grespectively) were markedly different from SBD-6H. The K_(d) of SBD-6His approximately 2-fold smaller than that of Aspergillus SBDs, whereasthe B_(max) of SBD-6H is markedly about 70-fold higher than that ofAspergillus SBDs These results indicated that both the starch-bindingaffinity and capacity of SBD-6H are grater than Aspergillus SBDs.

Example 5 (A) Construction of SBD-eGFP

The eGFP gene fragment was subsequently ligated into pET-SBD at NcoI andXhoI sites to generate pSBD-eGFP. The plasmid construct was as shown inFIG. 5. SBD-eGFP encoded by pSBD-eGFP contains a N-terminal SBD-6H and aC-terminal eGFP. The construct was transformed into competentEscherichia coli BL21-Gold (DE3) (Novagen) for protein expression.

(B) Expression and Purification of SBD-eGFP

BL21-Gold (DE3) cells transformed with pET-SBD were grown in LB mediumcontaining 100 μg/ml ampicillin at 37° C. until the OD₆₀₀ reached 0.6.The temperature was then reduced to 20° C., and isopropylβ-D-thiogalactoside (IPTG) was added to a final concentration of 400 μMto induce recombinant protein expression. After further incubation for16 h, cells were harvested by centrifugation at 3,700 g for 15 min at 4°C., the resultant pellet was resuspended in 20 ml of binding buffer (50mM sodium acetate, pH 5.5) and then homogenized (EmulsiFlex-05homogenizer). The cell debris was removed by centrifugation at 16,000 gfor 15 min at 4° C. The supernatant was applied directly to the granularcorn starch (pre-washed with binding buffer) and then incubated at 25°C. for 30 min with gentle shaking. The starch was washed with 10 columnvolumes of binding buffer twice and then eluted with 2 column volumes ofelution buffer (50 mM glycine/NaOH, pH 10). The solution was dialyzedagainst binding buffer employing an Amicon stirred-cell concentrator(Millipore) equipped with a PM-10 membrane (10 kDa cut-off). Eachfraction was analyzed by 15% SDS-PAGE gel and then stained withCoomassie brilliant blue. The protein concentrations were assayed bybicinchoninic acid (BCA) reagent kit (Pierce).

Purified SBD-eGFP was analyzed by 15% SDS-PAGE and stained withCoomassie brilliant blue as shown in FIG. 6.

1. A starch binding domain characterized by having an amino acidsequence selected from SEQ ID NO. 2 or SEQ ID NO.
 3. 2. The starchbinding domain of claim 1, which is obtainable from Rhizopus spp.
 3. Thestarch binding domain of claim 2, wherein the Rhizopus spp is Rhizopusoryzae.
 4. A kit for purifying a recombinant protein comprises anexpression vector, wherein the expression vector comprises a geneencoding starch binding domain selected from SEQ ID NO. 1, SEQ ID NO. 2or SEQ ID NO.
 3. 5. The kit of claim 4, which further comprises anaffinity matrix to bind the starch binging domain.
 6. The kit of claim4, which further comprises an elution buffer used to separate therecombinant protein and the affinity matrix.
 7. The kit of claim 4,wherein the recombinant protein is an antibody, an antigen, atherapeutic compound, or an enzyme.
 8. The kit of claim 5, wherein theaffinity matrix is selected from the groups consisting of: mannose,starch, dextran, glucan, and a molecule containing glucose-glucoselinkage structure.
 9. The kit of claim 8, wherein the elution buffer isselected from the groups consisting of acid, alkaline, salt, and sugar.10. The kit of claim 4, wherein the recombinant protein is used fordestroy/detection of pathogen, vaccine, or oral care composition. 11.The kit of claim 10, wherein the oral care composition is selected fromthe group consisting of a toothpaste, dental cream, gel or tooth powder,odontic, mouthish, pre- or post brushing rinse formulation, chewing gum,lozenge, and candy.
 12. The kit of claim 11, wherein the oral carecomposition is further comprises a fusion product between one or moreSBDs and an enzyme selected from the group consisting of oxidases,peroxidases, proteases, lipases, glycosidases, lipases, esterases,deaminases, ureases and polysaccharide hydrolases.
 13. A method ofsorting carbohydrate-containing molecule in a sample containing variouscarbohydrate-containing molecules comprises: (a) preparing a set ofseparator with different K_(d) values of carbohydrate binding domain;(b) putting the sample into the set of separator; and (c) sorting themolecule into bound and unbound groups in accordance with differentK_(d) values.
 14. The method of claim 13, wherein the carbohydrate isselected from the groups consisting of monosaccharide, disaccharide, andpolysaccharide.
 15. The method of claim 14, wherein the carbohydrate isa glycoprotein.
 16. The method of claim 13, wherein the separator is acolumn with gradient K_(d) value for starch.
 17. The method of claim 13,wherein the sorting step includes eluting the molecules and collectingthe unbound and bound molecules in different container.
 18. The methodof claim 17, wherein the eluted molecules could be separated based onphysical and chemical properties.
 19. The method of claim 18, whereinthe properties are selected from the groups consisting of liquidchromatography analysis, mass spectrometric analysis, lectinimmunosensor techniques, two-dimensional gel electrophoresis,enzymatical techniques, and chemical derivatizations
 20. The method ofclaim 13, wherein the molecules include the molecules of interest. 21.The method of claim 20, wherein the molecules of interest areglycoproteins.
 22. The method of claim 13, wherein the carbohydratebinding domain is starch binding domain.
 23. The method of claim 22,wherein the starch binding domain is obtained from the groups consistingof Rhizopus, Arxula, Lipomyces, Aspergillus, Bacillus, Clostridium,Cryptococcus, Fusarium, Geobacillus, Neurospora, Pseudomonas,Streptomyces, Thielavia, and Thermoanaerobacter.
 24. The methodaccording to claim 23, wherein the Rhizopus is Rhizopus oryzae ormutants thereof.